Methods for assessing the purity of a mesenchymal stem cells preparation

ABSTRACT

The present invention relates to a method for assessing, evaluating and/monitoring the purity of a mesenchymal stem cells preparation, in particular of an adipose stem cells preparation, comprising measuring the expression level of at least one growth factor.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/571,461, filed Nov. 2, 2017, which is a 35 U.S.C. § 371 filing ofInternational Patent Application No. PCT/EP2016/060351, filed May 9,2016, which claims priority to U.S. Provisional Patent Application Ser.No. 62/158,875, filed May 8, 2015, and European Patent Application No.15179417.9, filed Jul. 31, 2015. Each of the aforementioned applicationsis incorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention relates to methods for assessing the purity of acell preparation, in particular of a mesenchymal stem cells preparation.In particular, the present invention relates to growth factors asbiomarker of the purity of a mesenchymal stem cells preparation.

BACKGROUND OF INVENTION

The use of stem cell-based therapies for the repair and regeneration ofvarious tissues and organs offers alternative therapeutic solutions fora number of diseases. Mesenchymal stem cells (MSC) are plastic-adherentstromal cells characterized by their ability to differentiate intomesenchymal tissues such as bone, cartilage and fat. Because of theseproperties, MSC seem to be an ideal population of stem cells forpractical regenerative medicine. One of the richest sources of MSC isadipose tissue and adipose-tissue derived stem cells (ASC) are widelyinvestigated for development of new therapies in the field ofregenerative medicine (such as, for example, for wound healing,bone/cartilage regeneration, Crohn's disease, . . . ).

Cell therapy products must be manufactured following the “GoodManufacturing Practice” recommendations which required a purity analysisof the final cellular products. However, fibroblasts may be common cellcontaminants that affect the purity of mesenchymal stem cellpreparations. Currently, cell therapy products remain non-characterizedin term of cellular purity for fibroblastic cells. Indeed, currentdefined criteria for mesenchymal stem cells, including ASC,characterization are (i) plastic adherence for cell spreading andproliferation; (ii) surface markers profile (CD44+, CD45−, CD73+, CD90+,CD105+); and (iii) differentiation capacity towardadipose/osteogenic/chondrogenic lineages. However, mesenchymal stemcells and fibroblasts are considered nowadays as a subfamily without anycapacities to differentiate according to these criteria (Hematti,Cytotherapy, 2012; 14:516-521). Fibroblasts are ubiquitous cells,present in variable tissues (dermis, adipose tissue, muscle . . . ).They are also plastic-adherent, express the similar surface markerphenotype as MSC and have the capacity to differentiate towards themesenchymal lineages when cultivated in specific media.

Few studies relate to the identification of fibroblasts from othercells. Pilling et al. discloses the identification of markers thatdiscriminate between human peripheral blood monocytes, tissuemacrophages, fibrocytes and fibroblasts (PLoS One. 2009, 4(10):e7475).However mesenchymal stem cells are not included in this study. MoreoverGoodpaster et al. describes that fibroblasts may be positivelyidentified by the TE-7 antibody which specifically recognizes growingand quiescent fibroblasts in formalin-fixed, paraffin-embedded tissuesamples (Journal of Histochemistry & Cytochemistry. 2008,56(4):347-358). Nevertheless the TE-7 antibody was not tested onmesenchymal stem cells.

Accordingly, there is currently no quantitative and objective method todistinguish MSC from fibroblasts. Fibroblasts, however, are associatedwith cancer cells at all stages of cancer progression. A cell therapyproduct comprising fibroblasts is thus potentially carcinogenic. Ittherefore exists a need of tools to assess the purity of an MSCpreparation with respect to fibroblastic contamination, in particular incontext of cell therapy products preparation.

The inventors herein surprisingly demonstrate that mesenchymal stemcells and fibroblasts can be differentiated according to their capacityto secrete factors such as SDF-1α and VEGF.

Therefore, the present invention relates to a method for assessing thepresence of fibroblasts in a preparation of mesenchymal stem cells, e.g.a composition comprising cells, and for quantifying the purity of such apreparation.

SUMMARY

This invention relates to an in vitro method for assessing, evaluatingand/or monitoring the purity of a cell preparation comprisingmesenchymal stem cells (MSC), wherein said method comprises measuringthe expression level of at least one growth factor expressed by saidcell preparation, wherein said at least one growth factor is SDF-1αand/or VEGF.

According to one embodiment, the method of the invention furthercomprises comparing the measured expression level with a referenceexpression level.

In one embodiment, the mesenchymal stem cells of the cell preparation ofthe invention are isolated from tissues selected from the groupcomprising adipose tissue, bone marrow, umbilical cord blood, amnioticfluid, Wharton's jelly, placenta, peripheral blood, fallopian tube,corneal stroma, lung, muscle and fetal liver. In a particularembodiment, the mesenchymal stem cells are adipose stem cells (ASC).

According to one embodiment, the expression level of at least one growthfactor expressed by said cell preparation of the invention is assessedat the protein level, preferably by the detection and/or quantificationof said at least one growth factor secreted in the cell culturesupernatant. In a particular embodiment, the expression level isassessed at the RNA level, preferably by RT-PCR, RT-qPCR, Northern Blotand/or hybridization techniques.

According to one embodiment, the cell preparation of the invention issubstantially pure when the SDF-1α expression level is of at most 100pg/ml, preferably of at most 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.

According to another embodiment, the cell preparation of the inventionis substantially pure when the SDF-1α expression level is of at most 100pg/ml, preferably of at most 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and/or the VEGFexpression level is of at least 200 pg/ml in the cell culture medium,preferably of at least 250, 260, 270, 280, 281, 282, 283, 284, 285, 286,287, 288, 289 or 290 pg/ml; wherein said cell preparation is cultured inhypoxic conditions, preferably at about 0.1% O₂, and at highconcentration of glucose, preferably at about 4.5 g/l of glucose, beforemeasuring the expression level.

According to another embodiment, the cell preparation of the inventionis substantially pure when the SDF-1α expression level is of at most 100pg/ml, preferably of at most 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and/or the VEGFexpression level is of at least 90 pg/ml in the cell culture medium,preferably of at least 95, 100, 105, 110, 111, 112, 113, 114, 115, 116,117, 118, 119 or 120 pg/ml, wherein said cell preparation is cultured attissular oxygen tension, preferably at about 5% O₂, and at highconcentration of glucose, preferably at about 4.5 g/l of glucose, beforemeasuring the expression level.

In one embodiment, the method of the invention is for assessing thequality or purity of a cell preparation comprising mesenchymal stemcells (MSC), wherein said cell preparation comprising MSC is to be usedas MSC-based cell therapy product in regenerative medicine.

This invention also relates to the use of a growth factor, preferablySDF-1α and/or VEGF, as a biomarker of the quality of a cell preparationcomprising mesenchymal stem cells (MSC), in particular of a cellpreparation comprising MSC to be used as MSC-based cell therapy productin regenerative medicine.

Another object of the invention is a cell population identified by thein vitro method as described hereinabove.

This invention also relates to a substantially pure mesenchymal stemcells population, preferably an adipose stem cells population.

This invention also encompasses to a kit for implementing the in vitromethod as described hereinabove, wherein said kit comprises means fordetermining or measuring the expression level of at least one growthfactor, optionally further comprising the reference for comparing theexpression level of said at least one growth factor.

Definitions

In the present invention, the following terms have the followingmeanings:

-   -   “Purity of a cell preparation” refers to the enrichment of cells        of interest from a heterogeneous population (also called mixed        population). In one embodiment, cells of the invention are        mesenchymal stem cells, preferably adipose tissue mesenchymal        stem cells. In one embodiment, the purity according to the        invention is expressed in percentage of mesenchymal stem cells,        preferably adipose tissue mesenchymal stem cells, from a mixed        population comprising other cell types, preferably fibroblasts.    -   “Mesenchymal stem cells” or MSCs, are multipotent stem cells        that can differentiate into a variety of cell types including:        osteogenic, chondrogenic, adipogenic, myelosupportive stroma,        myogenic, or neurogenic lineages. Mesenchymal stem cells can be        isolated from tissues including, without limitation, adipose        tissue, bone-marrow, umbilical cord tissue, amniotic fluid,        Wharton's jelly, placenta, peripheral blood, fallopian tube,        corneal stroma, lung, muscle, skin, bone, dental tissue,        pre-menstrual fluid, foreskin and fetal liver, and the like.    -   “Adipose tissue” refers to any fat tissue. The adipose tissue        may be brown, yellow or white adipose tissue. Preferably, the        adipose tissue is subcutaneous white adipose tissue. Adipose        tissue includes adipocytes and stroma. Adipose tissue may be        found throughout the body of an animal. For example, in mammals,        adipose tissue may be present in the omentum, bone marrow,        subcutaneous space, fat pads (e.g., scapular or infrapatellar        fat pads), and surrounding most organs. Cells obtained from        adipose tissue may comprise a primary cell culture or a        progenitor cell line. The adipose tissue may be from any        organism having fat tissue.    -   “Adipose tissue-derived cell” refers to a cell that originates        from adipose tissue. In particular, “adipose tissue mesenchymal        stem cells” (ASC) refer to stromal cells that originate from        adipose tissue which can serve as precursors to a variety of        different cell types such as, but not limited to, adipocytes,        osteocytes, chondrocytes.    -   “Growth factor” refers to any substance participating in the        regulation of the cellular proliferation or differentiation.    -   “Tissular oxygen levels” refers to oxygen levels from about 3%        to about 6%, preferably at an oxygen level of about 5%.    -   “Hypoxic environment” refers to oxygen levels from about 0% to        about 1%, preferably at an oxygen level of about 0.1%.    -   “Normoglycemic conditions” refers to a concentration of glucose        of from about 0.5 g/l to about 1.5 g/l, preferably at a        concentration of glucose of about 1 g/1.    -   “Hyperglycaemic conditions” refers to a concentration of glucose        from about 2 g/l to about 10 g/l, preferably from about 3 g/l to        about 6 g/l, more preferably at a concentration of glucose of        about 4.5 g/l.    -   “About” preceding a value means plus or less 10% of said value.    -   “Passaging”, also known as subculture or splitting cells, refers        to transferring a small number of cells into a new vessel when        cells are at confluence or almost, to prolong the life and/or        expand the number of cells in the culture. In one embodiment,        the passage 0 (P0) is the point at which cells were initially        placed in culture.    -   “Late passaged mesenchymal stem cell” refers to a cell        exhibiting a less immunogenic characteristic when compared to an        earlier passaged cell. The immunogenicity of a mesenchymal stem        cell corresponds to the number of passages. Preferably, the cell        has been passaged up to at least the fourth passage, more        preferably, the cell has been passaged up to at least the sixth        passage, and most preferably, the cell has been passaged up to        at least the eight passage.

DETAILED DESCRIPTION

This invention relates to a method, preferably an in vitro method, forassessing, evaluating and/or monitoring the purity of a cell preparationcomprising mesenchymal stem cells (MSC), e.g. a composition of cellscomprising MSC, wherein said method comprises determining or measuringthe expression level of at least one growth factor expressed by saidcell preparation. In one embodiment, MSC are adipose tissue-derivedmesenchymal stem cells (ASC).

In one embodiment, the method of the invention is for assessing thepresence of fibroblasts in a cell preparation comprising mesenchymalstem cells. According to this embodiment, the method of the inventionmay thus correspond to a quality control method, aiming at checking, forexample, the purity of a cell preparation comprising MSC with respect tofibroblastic contamination. Those MSC may be, for example, used forMSC-based cell therapy product.

In one embodiment, the method of the invention is for quantifying thepurity of a cell preparation comprising mesenchymal stem cells. In oneembodiment, the cell preparation is a heterogeneous preparationcomprising mesenchymal stem cells and other cell types. In a particularembodiment, the cell preparation comprises mesenchymal stem cells andfibroblasts.

In one embodiment, the method of the invention is for quantifying thepercentage of mesenchymal stem cells from a heterogeneous populationcomprising mesenchymal stem cells and other cell types, preferablyfibroblasts.

The Applicant demonstrated that with progressive contaminations of MSCby fibroblasts, the expression level of growth factors tends to increaseor decrease. Consequently, this invention demonstrates the capacity todiscriminate MSC from fibroblasts, based on their specific growthfactors expression. In particular, the discrimination may be based ontheir growth factors expression in specific conditions of oxygenationand/or glycemia.

Therefore, in one embodiment, the method of the invention is forassessing, evaluating and/or monitoring the purity of a cell preparationcomprising MSC with respect to fibroblastic contamination, based ontheir specific growth factors expression.

Examples of growth factors include, but are not limited to,adipocytokines, angiopoietins, angiopoietin-like proteins and receptorsthereof, chemokines and receptors thereof, common beta chain receptors,common gamma chain receptors, EGF, FGF, hedgehog proteins, IGF,interferons, interleukins and receptors, PDGF, TGF, TNF, VEGF, SDF-1 andWnt.

In one embodiment, the method of the invention comprises measuring theexpression level of SDF-1 (stromal cell-derived factor 1, also known asC-X-C motif chemokine 12 or CXCL12, Pre-B Cell Growth-stimulating Factor(PBSF), SCYB12 or TLSF) and/or VEGF (vascular endothelial growthfactor). Preferably, SDF-1 is in the form of SDF-1α.

In one embodiment, the method according to the invention comprisesmeasuring the expression level of SDF-1α. In another embodiment, themethod according to the invention comprises measuring the expressionlevel of VEGF. In another embodiment, the method according to theinvention comprises measuring the expression level of SDF-1α and VEGF.

According to one embodiment, MSC are isolated from tissues selected fromthe group comprising adipose tissue, bone marrow, umbilical cord blood,Wharton's jelly (such as, for example, Wharton's jelly found within theumbilical cord), placenta, peripheral blood, fallopian tube, cornealstroma, lung, muscle skin, bone, dental tissue and fetal liver, or thelike. In a particular embodiment, MSC are isolated from adipose tissue.In a preferred embodiment, MSC are adipose stem cells (ASC).

In one embodiment, MSC are isolated from any warm-blooded animaltissues, preferably from human tissues. In a particular embodiment, MSCare human ASC.

In one embodiment, the cells are cells in culture, preferably are celllines and/or are derived from primary cells, i.e. cells isolatedstraight from the tissue. In one embodiment, the cells are recoveredfrom a sample from an individual, obtained for example by biopsy.Preferably, the step of recovering the sample from an individual is notpart of the method of the present invention.

Isolation of mesenchymal stem cells may be accomplished by anyacceptable method known to one of ordinary skill in the art. Examples ofmethods for isolating MSC include, but are not limited to, digestion bycollagenase, trypsinization, or explant culture.

In a particular embodiment, mesenchymal stem cells are isolated fromadipose tissue by digestion of the tissue, for example by collagenase.

According to one embodiment of the invention, after isolation, the cellpreparation comprising MSC is cultured in any culture medium designed tosupport the growth of the cells known to one of ordinary skill in theart. As used herein, such culture medium is called “proliferationmedium” or “growth medium”. Examples of growth medium include, withoutlimitation, MEM, DMEM, IMDM, RPMI 1640, FGM or FGM-2, 199/109 medium,HamF10/HamF12 or McCoy's 5A, preferably DMEM or RPMI.

In one embodiment, the growth medium may further comprise anysupplementary factors known by the person skilled in the art that may beused in cell culture. Examples of supplementary factors include, but arenot limited to, FBS; glycine; amino acids, such as glutamine,asparagine, glutamic acid, aspartic acid, serine, proline or alanine,preferably the L-configuration of amino acids; and antibiotics, such asstreptomycin or penicillin.

In a particular embodiment, the cell preparation comprising MSC iscultured in DMEM supplemented with fetal bovine serum, glutamine,preferably L-glutamine, and antibiotics such as penicillin, streptomycinand amphotericin B.

In one embodiment, the cell preparation comprising MSC may becontaminated by other types of cells, such as, for example, byfibroblasts. In a particular embodiment, the cell preparation comprisingMSC is contaminated by fibroblasts.

In one embodiment, the cell preparation comprising MSC is cultured ingrowth medium up to at least 2 passages, preferably at least 3 passages,more preferably at least 4 passages. As used herein, the term “culturedup to at least 4 passages” means that the cell preparation is detachedand transferred into a new vessel up to at least 4 times. In oneembodiment, the mesenchymal stem cells of the cell preparation are latepassaged mesenchymal stem cells.

For passaging cells, cells may be detached by one of several methodsknown to one of ordinary skill in the art, including trypsin treatmentto break down the proteins responsible for surface adherence, chelatingsodium ions with EDTA which disrupts some protein adherence mechanisms,or mechanical methods like repeated washing or use of a cell scraper.The detached cells are then resuspended in fresh medium.

In one embodiment, the cell preparation comprising MSC is cultured forat least 24 hours, preferably for at least 36, 48, 60 or 72 hours. Inanother embodiment, the cell preparation comprising MSC is cultured forat least 1 day, preferably for at least 2, 3, 4, 5, 6 or 7 days. Inanother embodiment, the cell preparation comprising MSC is cultured forat least 10, 15, 20, 25, 30, 35 or 40 days.

According to one embodiment, the cell preparation comprising MSC iscultured in standard culture conditions. As used herein, “standardculture conditions” means at a temperature of about 37° C., and at atension of about 21% O₂ and of about 5% CO₂.

In one embodiment, the step of culturing the cell preparation comprisingMSC is not part of the method of the present invention.

In one embodiment, culture conditions of the cell preparation comprisingMSC are changed before measuring the expression level of at least onegrowth factor.

As used herein, the term “before measuring the expression level of atleast one growth factor” means at least 6 hours, preferably at least 9,12, 15, 18 or 24 hours between the last passage and the measurement ofthe expression level of at least one growth factor.

According to one embodiment, the cell preparation comprising MSC iscultured at an oxygen tension of no more than 21%, preferably at most of15%, more preferably at most of 10% before measuring the expressionlevel of at least one growth factor. In a particular embodiment, thecell preparation comprising MSC is cultured at an oxygen level fromabout 3% to about 6%, preferably at about 5% O₂ corresponding totissular oxygen tension before measuring the expression level of atleast one growth factor. In another embodiment, the cell preparationcomprising MSC is cultured at an oxygen level from about 0% to about 1%,at about 0.1% O₂ corresponding to hypoxic environment before measuringthe expression level of at least one growth factor.

According to another embodiment, the cell preparation comprising MSC iscultured in a medium comprising from about 0.1 to about 10 g/l ofglucose, preferably from about 0.5 to about 6 g/l of glucose, morepreferably from about 1 to about 4.5 g/l of glucose before measuring theexpression level of at least one growth factor.

In one embodiment, the cell preparation comprising MSC is cultured in amedium comprising a low concentration of glucose, corresponding tonormal blood sugar levels in vivo, i.e. in a medium comprising fromabout 0.5 to about 1.5 g/l of glucose, preferably about 1 g/l of glucosebefore measuring the expression level of at least one growth factor. Inanother embodiment, the cell preparation comprising MSC is cultured in amedium comprising a high concentration of glucose, corresponding tohyperglycaemic conditions, i.e. in a medium comprising from about 2 toabout 10 g/l of glucose, preferably from about 3 to about 6 g/l ofglucose, more preferably about 4.5 g/l of glucose before measuring theexpression level of at least one growth factor.

In one embodiment, the cell preparation comprising MSC is cultured atabout 21% O₂ and about 1 g/l glucose before measuring the expressionlevel of at least one growth factor. In another embodiment, the cellpreparation comprising MSC is cultured at about 21% O₂ and about 4.5 g/lglucose before measuring the expression level of at least one growthfactor. In another embodiment, the cell preparation comprising MSC iscultured at about 5% O₂ and about 1 g/l glucose before measuring theexpression level of at least one growth factor. In another embodiment,the cell preparation comprising MSC is cultured at about 5% O₂ and about4.5 g/l glucose before measuring the expression level of at least onegrowth factor. In another embodiment, the cell preparation comprisingMSC is cultured at about 0.1% O₂ and about 1 g/l glucose beforemeasuring the expression level of at least one growth factor. In anotherembodiment, the cell preparation comprising MSC is cultured at about0.1% O₂ and about 4.5 g/l glucose before measuring the expression levelof at least one growth factor.

According to another embodiment, culture conditions of the cellpreparation comprising MSC are always the same during all steps of themethod of the invention.

In one embodiment of the invention, the method comprises the followingsteps:

-   -   a) culturing a cell preparation comprising mesenchymal stem        cells,    -   b) optionally changing the culture conditions, and    -   c) quantifying expression level of at least one growth factor,        preferably SDF-1α and/or VEGF.

In a preferred embodiment, the method of the invention for assessing thepurity of a cell preparation comprising mesenchymal stem cells,preferably adipose stem cells, comprises the following steps:

-   -   a) culturing a cell preparation comprising mesenchymal stem        cells, preferably adipose stem cells,    -   b) optionally changing the culture conditions, preferably the O₂        tension, and    -   c) quantifying SDF-1α and/or VEGF expression level.

In a particular embodiment, the method of the invention for assessingthe purity of a cell preparation comprising mesenchymal stem cells,preferably adipose stem cells, comprises the following steps:

-   -   a) culturing a cell preparation comprising mesenchymal stem        cells, preferably adipose stem cells,    -   b) optionally changing the culture conditions, preferably the O₂        tension, and    -   c) quantifying SDF-1α expression level.

In a particular embodiment, the method of the invention for assessingthe purity of a cell preparation comprising mesenchymal stem cells,preferably adipose stem cells, comprises the following steps:

-   -   a) culturing a cell preparation comprising mesenchymal stem        cells, preferably adipose stem cells,    -   b) optionally changing the culture conditions, preferably the O₂        tension, and    -   c) quantifying SDF-1α and VEGF expression level.

As used herein, the term “expression” may refer alternatively to thetranscription of a growth factor (i.e. expression of the RNA) or to thetranslation (i.e. expression of the protein) of a growth factor, or tothe presence of the growth factor in the supernatant of the cells inculture (i.e. to the secretion of the growth factor).

Methods for determining the expression level are well-known from theskilled artisan, and include, without limitation, determining thetranscriptome (in an embodiment wherein expression relates totranscription of a growth factor) or proteome (in an embodiment whereinexpression relates to translation or secretion of a growth factor) of acell.

In one embodiment of the invention, the expression of the growth factoris assessed at the RNA level. Methods for assessing the transcriptionlevel of a growth factor are well known in the prior art. Examples ofsuch methods include, but are not limited to, RT-PCR, RT-qPCR, NorthernBlot, hybridization techniques such as, for example, use of microarrays,and combination thereof including but not limited to, hybridization ofamplicons obtained by RT-PCR, sequencing such as, for example,next-generation DNA sequencing (NGS) or RNA-seq (also known as “WholeTranscriptome Shotgun Sequencing”) and the like.

In another embodiment of the invention, the expression of the growthfactor is assessed at the protein level. Methods for determining aprotein level in a sample are well-known in the art. Examples of suchmethods include, but are not limited to, immunohistochemistry, Multiplexmethods (Luminex), western blot, enzyme-linked immunosorbent assay(ELISA), sandwich ELISA, fluorescent-linked immunosorbent assay (FLISA),enzyme immunoassay (EIA), radioimmunoassay (RIA), flow cytometry (FACS)and the like. Preferably determining the protein level of the growthfactor is assessed by enzyme-linked immunosorbent assay (ELISA).

In one embodiment of the invention, determining the expression level ofthe growth factor specifically corresponds to the detection andquantification of said growth factor secreted in the cell culturesupernatant. In a particular embodiment, the method of the inventioncomprises measuring expression level of SDF-1α and/or VEGF in the cellculture supernatant of the cell preparation comprising MSC.

In one embodiment, the step of measuring the expression level of atleast one growth factor is performed when the MSC comprised in the cellpreparation are at a density leading to about 80% confluence, preferablyto about 85, 90, 95, 99 or 100% confluence.

In the meaning of the invention, the cell preparation comprising MSC issubstantially pure when said cell preparation comprises less than 25% offibroblasts, preferably less than 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12 or 11% of fibroblasts. In one embodiment, the cellpreparation comprising MSC is substantially pure when said cellpreparation comprises less than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% offibroblasts.

According to one embodiment, the cell preparation comprising MSC issubstantially pure when the SDF-1α expression level of the cellpreparation comprising MSC is at most of 100 pg/ml, preferably at mostof 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 pg/ml.

In one embodiment, the cell preparation comprising MSC is substantiallypure when the SDF-1α expression level of the cell preparation comprisingMSC cultured at about 21% 02, is at most of 50 pg/ml, preferably at mostof 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2 or 1 pg/ml.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the SDF-1α expression level of the cellpreparation comprising MSC cultured at tissular oxygen tension,preferably at about 5% O₂, is at most of 50 pg/ml, preferably at most of40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 pg/ml.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the SDF-1α expression level of the cellpreparation comprising MSC cultured in hypoxic condition, preferably atabout 0.1% O₂, is at most of 50 pg/ml, preferably at most of 40, 30, 25,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1pg/ml.

In one embodiment, the cell preparation comprising MSC is substantiallypure when the SDF-1α expression level of the cell preparation comprisingMSC cultured at low concentration of glucose, preferably at about 1 g/lof glucose, is at most of 100 pg/ml, preferably at most of 50, 40, 30,25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2or 1 pg/ml.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the SDF-1α expression level of the cellpreparation comprising MSC cultured at high concentration of glucose,preferably at about 4.5 g/l of glucose, is at most of 50 pg/ml,preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.

In one embodiment, the cell preparation comprising MSC is substantiallypure when the SDF-1α expression level of the cell preparation comprisingMSC cultured at about 21% O₂ and at low concentration of glucose,preferably at about 1 g/l of glucose, is at most of 100 pg/ml,preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the SDF-1α expression level of the cellpreparation comprising MSC cultured at about 21% O₂ and at highconcentration of glucose, preferably at about 4.5 g/l of glucose, is atmost of 50 pg/ml, preferably at most of 40, 30, 25, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the SDF-1α expression level of the cellpreparation comprising MSC cultured at tissular oxygen tension,preferably at about 5% O₂, and at low concentration of glucose,preferably at about 1 g/l of glucose, is at most of 100 pg/ml,preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the SDF-1α expression level of the cellpreparation comprising MSC cultured at tissular oxygen tension,preferably at about 5% O₂, and at high concentration of glucose,preferably at about 4.5 g/l of glucose, is at most of 50 pg/ml,preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the SDF-1α expression level of the cellpreparation comprising MSC cultured in hypoxic condition, preferably atabout 0.1% O₂, and at low concentration of glucose, preferably at about1 g/l of glucose, is at most of 100 pg/ml, preferably at most of 50, 40,30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 pg/ml.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the SDF-1α expression level of the cellpreparation comprising MSC cultured in hypoxic condition, preferably atabout 0.1% O₂, and at high concentration of glucose, preferably at about4.5 g/l of glucose, is at most of 50 pg/ml, preferably at most of 40,30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 pg/ml.

According to one embodiment, the cell preparation comprising MSC issubstantially pure when the VEGF expression level of the cellpreparation comprising MSC cultured in hypoxic conditions, preferably atabout 0.1% O₂, and at high concentration of glucose, preferably at about4.5 g/l of glucose, is at least of 200 pg/ml, preferably at least of250, 260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 or 290pg/ml.

According to another embodiment, the cell preparation comprising MSC issubstantially pure when the VEGF expression level of the cellpreparation comprising MSC cultured at tissular oxygen tension,preferably at about 5% O₂, and at high concentration of glucose,preferably at about 4.5 g/l of glucose, is at least of 90 pg/ml,preferably at least of 95, 100, 105, 110, 111, 112, 113, 114, 115, 116,117, 188, 119 or 120 pg/ml.

According to another embodiment, the cell preparation comprising MSC issubstantially pure when the VEGF expression level of the cellpreparation comprising MSC cultured tissular oxygen tension, preferablyat about 5% O₂, and at low concentration of glucose, preferably at about1 g/l of glucose, is at least of 160 pg/ml, preferably at least of 161,162, 163, 164, 165, 166, 167, 168 or 169 pg/ml, more preferably at leastof 170 pg/ml.

In one embodiment, the cell preparation comprising MSC is substantiallypure when the cell preparation comprising MSC cultured in hypoxiccondition, preferably at about 0.1% 02, and at high concentration ofglucose, preferably at about 4.5 g/l of glucose, presents a SDF-1αexpression level of at most 50 pg/ml, preferably of at most 40, 30, 25,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1pg/ml; and a VEGF expression level of at least 200 pg/ml, preferably ofat least 250, 260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289or 290 pg/ml.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the cell preparation comprising MSC cultured attissular oxygen tension, preferably at about 5% O₂, and at highconcentration of glucose, preferably at about 4.5 g/l of glucose,presents a SDF-1α expression level of at most 50 pg/ml, preferably of atmost 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2 or 1 pg/ml; and a VEGF expression level of at least 90 pg/ml,preferably of at least 95, 100, 105, 110, 111, 112, 113, 114, 115, 116,117, 188, 119 or 120 pg/ml.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the cell preparation comprising MSC cultured attissular oxygen tension, preferably at about 5% O₂, and at lowconcentration of glucose, preferably at about 1 g/l of glucose, presentsa SDF-1α expression level of at most 100 pg/ml, preferably of at most50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2 or 1 pg/ml; and a VEGF expression level of at least 160pg/ml, preferably at least of 161, 162, 163, 164, 165, 166, 167, 168 or169 pg/ml, more preferably at least of 170 pg/ml.

In one embodiment of the invention, the method comprises the followingsteps:

-   -   a) culturing a cell preparation comprising mesenchymal stem        cells,    -   b) optionally changing the culture conditions, and    -   c) quantifying secretion of at least one growth factor in the        cell culture supernatant, preferably SDF-1α and/or VEGF.

In a preferred embodiment, the method of the invention for assessing thepurity of a cell preparation comprising mesenchymal stem cells,preferably adipose stem cells, comprises the following steps:

-   -   a) culturing a cell preparation comprising mesenchymal stem        cells, preferably adipose stem cells,    -   b) optionally changing the culture conditions, preferably the O₂        tension, and    -   c) quantifying SDF-1α and/or VEGF secretion in the cell culture        supernatant.

In a particular embodiment, the method of the invention for assessingthe purity of a cell preparation comprising mesenchymal stem cells,preferably adipose stem cells, comprises the following steps:

-   -   a) culturing a cell preparation comprising mesenchymal stem        cells, preferably adipose stem cells,    -   b) optionally changing the culture conditions, preferably the O₂        tension, and    -   c) quantifying SDF-1α secretion in the cell culture supernatant.

In a particular embodiment, the method of the invention for assessingthe purity of a cell preparation comprising mesenchymal stem cells,preferably adipose stem cells, comprises the following steps:

-   -   a) culturing a cell preparation comprising mesenchymal stem        cells, preferably adipose stem cells,    -   b) optionally changing the culture conditions, preferably the O₂        tension, and    -   c) quantifying SDF-1α and VEGF secretion in the cell culture        supernatant.

In one embodiment, the method of the invention further comprises a stepof comparing the measured expression level with a reference level.

As used herein, the term “reference” broadly encompasses any suitablereference expression level which may be used as a basis for comparisonwith respect to the measured expression level.

In one embodiment, the reference is a pure fibroblasts preparation. Asused herein, “a pure fibroblasts preparation”, e.g. a compositioncomprising fibroblasts, means a preparation known to be free from anyother types of cells than fibroblasts. According to one embodiment, thepure fibroblasts preparation is cultured in the same conditions as thecell preparation comprising MSC.

In one embodiment, the cell preparation comprising MSC is substantiallypure when the SDF-1α expression level of the cell preparation comprisingMSC is significatively lower than the SDF-1α expression level of thepure fibroblasts preparation. As used herein, the term “significativelylower” means at least 1.5 fold lower, preferably at least 2, 3 or 4 foldlower, more preferably at least 5, 6, 7 or 8 fold lower.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the VEGF expression level of the cellpreparation comprising MSC is significatively higher than the VEGFexpression level of the pure fibroblasts preparation. As used herein,the term “significatively higher” means at least 1.5 fold higher,preferably at least 2 fold higher, more preferably at least 2.1, 2.2,2.3, 2.4 or 2.5 fold higher.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the SDF-1α expression level of the cellpreparation comprising MSC is significatively lower than the SDF-1αexpression level of the pure fibroblasts preparation and when the VEGFexpression level of the cell preparation comprising MSC issignificatively higher than the VEGF expression level of the purefibroblasts preparation.

In another embodiment, the reference sample is a pure mesenchymal stemcells preparation. As used herein, “a pure mesenchymal stem cellspreparation”, e.g. a composition comprising mesenchymal stem cells,means a preparation known to be free from fibroblasts. According to oneembodiment, the pure mesenchymal stem cells preparation is cultured inthe same conditions as the cell preparation comprising MSC to be tested.

In one embodiment, the cell preparation comprising MSC is substantiallypure when the SDF-1α expression level of the cell preparation comprisingMSC is at least 80%, preferably at least 85%, more preferably at least90%, 95%, 96%, 97%, 98%, 99% or 100% of the SDF-1α expression level ofthe pure MSC preparation.

In a particular embodiment, the cell preparation comprising MSC istotally pure, i.e. without any fibroblastic contamination, when theSDF-1α expression level of the cell preparation comprising MSC and ofthe pure MSC preparation are the same.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the VEGF expression level of the cellpreparation comprising MSC is at least 80%, preferably at least 85%,more preferably at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of theVEGF expression level of the pure cell preparation comprising MSC.

In a particular embodiment, the cell preparation comprising MSC istotally pure, i.e. without any fibroblastic contamination, when the VEGFexpression level of the cell preparation comprising MSC and of the pureMSC preparation are the same.

In another embodiment, the cell preparation comprising MSC issubstantially pure when the SDF-1α expression level of the cellpreparation comprising MSC is at least 80%, preferably at least 85%,more preferably at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of theSDF-1α expression level of the pure MSC preparation and when the VEGFexpression level of the cell preparation comprising MSC is at least 80%,preferably at least 85%, more preferably at least 90%, 95%, 96%, 97%,98%, 99% or 100% of the VEGF expression level of the pure MSCpreparation.

In one embodiment of the invention, the method comprises the followingsteps:

-   -   a) culturing a cell preparation comprising mesenchymal stem        cells,    -   b) optionally changing the culture conditions,    -   c) quantifying expression level of at least one growth factor,        preferably SDF-1α and/or VEGF, and    -   d) comparing the expression level measured in step (c) with a        reference expression level.

In a preferred embodiment, the method of the invention for assessing thepurity of a cell preparation comprising mesenchymal stem cells,preferably adipose stem cells, comprises the following steps:

-   -   a) culturing a cell preparation comprising mesenchymal stem        cells, preferably adipose stem cells,    -   b) optionally changing the culture conditions, preferably the O₂        tension,    -   c) quantifying SDF-1α and/or VEGF expression level, and    -   d) comparing the expression level measured in step (c) with        SDF-1α and/or VEGF expression levels of a reference population        of cells cultured in the same conditions as MSC.

In a particular embodiment, the method of the invention for assessingthe purity of a cell preparation comprising mesenchymal stem cells,preferably adipose stem cells, comprises the following steps:

-   -   a) culturing a cell preparation comprising mesenchymal stem        cells, preferably adipose stem cells,    -   b) optionally changing the culture conditions, preferably the O₂        tension,    -   c) quantifying SDF-1α and VEGF expression level, and    -   d) comparing the expression level measured in step (c) with        SDF-1α and VEGF expression levels of a reference population of        cells cultured in the same conditions as MSC.

The invention also relates to a cell population identified by the methodof the invention as described hereinabove.

In one embodiment, the cell population thus identified is assessed forits purity with respect to fibroblastic contamination.

In one embodiment, the cell population thus identified is substantiallypure, i.e. said cell population comprises less than 25% of fibroblasts,preferably less than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12or 11% of fibroblasts. In one embodiment, the cell preparation thusidentified of the invention comprises less than 10, 9, 8, 7, 6, 5, 4, 3,2 or 1% of fibroblasts.

According to one embodiment, the cell population thus identifiedpresents a SDF-1α expression level of at most 100 pg/ml, preferably atmost of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.

According to one embodiment, the cell population thus identifiedpresents a SDF-1α expression level of at most 50 pg/ml, preferably atmost of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 pg/ml when the cell preparation comprising MSC iscultured at about 21% O₂.

According to another embodiment, the cell population thus identifiedpresents a SDF-1α expression level of at most 50 pg/ml, preferably atmost of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 pg/ml when the cell preparation comprising MSC iscultured at tissular oxygen tension, preferably at about 5% O₂.

According to another embodiment, the cell population thus identifiedpresents a SDF-1α expression level of at most 50 pg/ml, preferably atmost of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 pg/ml when the cell preparation comprising MSC iscultured in hypoxic conditions, preferably at about 0.1% O₂.

According to one embodiment, the cell population thus identifiedpresents a SDF-1α expression level of at most 100 pg/ml, preferably atmost of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when the cell preparation comprising MSCis cultured at low concentration of glucose, preferably at about 1 g/lof glucose.

According to another embodiment, the cell population thus identifiedpresents a SDF-1α expression level of at most 50 pg/ml, preferably atmost of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 10 pg/ml when the cell preparation comprising MSC iscultured at high concentration of glucose, preferably at about 4.5 g/lof glucose.

In one embodiment, the cell population thus identified presents a SDF-1αexpression level of at most 100 pg/ml, preferably at most of 50, 40, 30,25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2or 1 pg/ml when the cell preparation comprising MSC is cultured at about21% O₂ and at low concentration of glucose, preferably at about 1 g/l ofglucose.

In another embodiment, the cell population thus identified presents aSDF-1α expression level of at most 50 pg/ml, preferably at most of 40,30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 pg/ml when the cell preparation comprising MSC is cultured atabout 21% O₂ and at high concentration of glucose, preferably at about4.5 g/l of glucose.

In one embodiment, the cell population thus identified presents a SDF-1αexpression level of at most 100 pg/ml, preferably at most of 50, 40, 30,25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2or 1 pg/ml when the cell preparation comprising MSC is cultured attissular oxygen tension, preferably at about 5% O₂, and at lowconcentration of glucose, preferably at about 1 g/l of glucose.

In another embodiment, the cell population thus identified presents aSDF-1α expression level of at most 50 pg/ml, preferably at most of 40,30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 pg/ml when the cell preparation comprising MSC is cultured attissular oxygen tension, preferably at about 5% O₂, and at highconcentration of glucose, preferably at about 4.5 g/l of glucose.

In one embodiment, the cell population thus identified presents a SDF-1αexpression level of at most 100 pg/ml, preferably at most of 50, 40, 30,25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2or 1 pg/ml when the cell preparation comprising MSC is cultured inhypoxic conditions, preferably at about 0.1% O₂, and at lowconcentration of glucose, preferably at about 1 g/l of glucose.

In another embodiment, the cell population thus identified presents aSDF-1α expression level of at most 50 pg/ml, preferably at most of 40,30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 pg/ml when the cell preparation comprising MSC is cultured inhypoxic conditions, preferably at about 0.1% O₂, and at highconcentration of glucose, preferably at about 4.5 g/l of glucose.

According to one embodiment, the cell population thus identifiedpresents a VEGF expression level of at least of 200 pg/ml, preferably atleast of 250, 260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289or 290 pg/ml when the cell preparation comprising MSC is cultured inhypoxic conditions, preferably at about 0.1% O₂, and high concentrationof glucose, preferably at about 4.5 g/l of glucose.

According to another embodiment, the cell population thus identifiedpresents a VEGF expression level of at least of 90 pg/ml, preferably atleast of 95, 100, 105, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119or 120 pg/ml when the cell preparation comprising MSC is cultured attissular oxygen tension, preferably at about 5% O₂, and highconcentration of glucose, preferably at about 4.5 g/l of glucose.

According to another embodiment, the cell population thus identifiedpresents a VEGF expression level of at least of 160 pg/ml, preferably atleast of 161, 162, 163, 164, 165, 166, 167, 168 or 169 pg/ml, morepreferably at least of 170 pg/ml when the cell preparation comprisingMSC is cultured at tissular oxygen tension, preferably at about 5% O₂,and low concentration of glucose, preferably at about 1 g/l of glucose.

In one embodiment, the cell population thus identified presents a SDF-1αexpression level of at most 50 pg/ml, preferably of at most 40, 30, 25,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1pg/ml; and a VEGF expression level of at least 200 pg/ml, preferably ofat least 250, 206, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289or 290 pg/ml, when the cell preparation comprising MSC is cultured inhypoxic conditions, preferably at about 0.1% O₂, and at highconcentration of glucose, preferably at about 4.5 g/l of glucose.

In another embodiment, the cell population thus identified presents aSDF-1α expression level of at most 50 pg/ml, preferably of at most 40,30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 pg/ml; and a VEGF expression level of at least 90 pg/ml,preferably of at least 95, 100, 105, 110, 111, 112, 113, 114, 115, 116,117, 118, 119 or 120 pg/ml, when the cell preparation comprising MSC iscultured at 5% O₂ and high concentration of glucose, preferably at about4.5 g/l of glucose.

In another embodiment, the cell preparation thus identified presents aSDF-1α expression level of at most 100 pg/ml, preferably of at most 50,40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 pg/ml; and a VEGF expression level of at least 160 pg/ml,preferably at least of 161, 162, 163, 164, 165, 166, 167, 168 or 169pg/ml, more preferably at least of 170 pg/ml, when the cell preparationcomprising MSC is cultured at 5% O₂ and low concentration of glucose,preferably at about 1 g/l of glucose.

Another object of the invention is a substantially pure mesenchymal stemcells population, preferably an adipose stem cells population.

In one embodiment, the substantially pure cell population of theinvention comprises less than 25% of fibroblasts, preferably less than24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 11% offibroblasts. In one embodiment, the cell preparation of the inventioncomprises less than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% of fibroblasts.

According to one embodiment, the substantially pure cell population ofthe invention presents a SDF-1α expression level of at most 100 pg/ml,preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml.

According to one embodiment, the substantially pure cell population ofthe invention presents a SDF-1α expression level of at most 50 pg/ml,preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when cultured at about 21% O₂.

According to another embodiment, the substantially pure cell populationof the invention presents a SDF-1α expression level of at most 50 pg/ml,preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when cultured at tissularoxygen tension, preferably at about 5% O₂.

According to another embodiment, the substantially pure cell populationof the invention presents a SDF-1α expression level of at most 50 pg/ml,preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when cultured in hypoxicconditions, preferably at about 0.1% O₂.

According to one embodiment, the substantially pure cell population ofthe invention presents a SDF-1α expression level of at most 100 pg/ml,preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when cultured at lowconcentration of glucose, preferably at about 1 g/l of glucose.

According to another embodiment, the substantially pure cell populationof the invention presents a SDF-1α expression level of at most 50 pg/ml,preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when cultured at highconcentration of glucose, preferably at about 4.5 g/l of glucose.

In one embodiment, the substantially pure cell population of theinvention presents a SDF-1α expression level of at most 100 pg/ml,preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when cultured at about 21%O₂ and at low concentration of glucose, preferably at about 1 g/l ofglucose.

In another embodiment, the substantially pure cell population of theinvention presents a SDF-1α expression level of at most 50 pg/ml,preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when cultured at about 21% O₂and at high concentration of glucose, preferably at about 4.5 g/l ofglucose.

In one embodiment, the substantially pure cell population of theinvention presents a SDF-1α expression level of at most 100 pg/ml,preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when cultured at tissularoxygen tension, preferably at about 5% O₂, and at low concentration ofglucose, preferably at about 1 g/l of glucose.

In another embodiment, the substantially pure cell population of theinvention presents a SDF-1α expression level of at most 50 pg/ml,preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when cultured at tissularoxygen tension, preferably at about 5% O₂, and at high concentration ofglucose, preferably at about 4.5 g/l of glucose.

In one embodiment, the substantially pure cell population of theinvention presents a SDF-1α expression level of at most 100 pg/ml,preferably at most of 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when cultured in hypoxicconditions, preferably at about 0.1% O₂, and at low concentration ofglucose, preferably at about 1 g/l of glucose.

In another embodiment, the substantially pure cell population of theinvention presents a SDF-1α expression level of at most 50 pg/ml,preferably at most of 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml when cultured in hypoxicconditions, preferably at about 0.1% O₂, and at high concentration ofglucose, preferably at about 4.5 g/l of glucose.

According to one embodiment, the substantially pure cell population ofthe invention presents a VEGF expression level of at least of 200 pg/ml,preferably at least of 250, 260, 270, 280, 281, 282, 283, 284, 285, 286,287, 288, 289 or 290 pg/ml when cultured in hypoxic conditions,preferably at about 0.1% O₂, and high concentration of glucose,preferably at about 4.5 g/l of glucose.

According to another embodiment, the substantially pure cell populationof the invention presents a VEGF expression level of at least of 90pg/ml, preferably at least of 95, 100, 105, 110, 111, 112, 113, 114,115, 116, 117, 118, 119 or 120 pg/ml when cultured at tissular oxygentension, preferably at about 5% O₂, and high concentration of glucose,preferably at about 4.5 g/l of glucose.

According to another embodiment, the substantially pure cell populationof the invention presents a VEGF expression level of at least of 160pg/ml, preferably at least of 161, 162, 163, 164, 165, 166, 167, 168 or169 pg/ml, more preferably at least of 170 pg/ml when cultured attissular oxygen tension, preferably at about 5% O₂, and lowconcentration of glucose, preferably at about 1 g/l of glucose.

In one embodiment, the substantially pure cell population of theinvention presents a SDF-1α expression level of at most 50 pg/ml,preferably of at most 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and a VEGF expression levelof at least 200 pg/ml, preferably of at least 250, 206, 270, 280, 281,282, 283, 284, 285, 286, 287, 288, 289 or 290 pg/ml, when cultured inhypoxic conditions, preferably at about 0.1% O₂, and high concentrationof glucose, preferably at about 4.5 g/l of glucose.

In another embodiment, the substantially pure cell population of theinvention presents a SDF-1α expression level of at most 50 pg/ml,preferably of at most 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and a VEGF expression levelof at least 90 pg/ml, preferably of at least 95, 100, 105, 110, 111,112, 113, 114, 115, 116, 117, 118, 119 or 120 pg/ml, when cultured attissular oxygen tension, preferably at about 5% O₂, and highconcentration of glucose, preferably at about 4.5 g/l of glucose.

In another embodiment, the substantially pure cell population of theinvention presents a SDF-1α expression level of at most 100 pg/ml,preferably of at most 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and a VEGF expressionlevel of at least 160 pg/ml, preferably at least of 161, 162, 163, 164,165, 166, 167, 168 or 169 pg/ml, more preferably at least of 170 pg/ml,when cultured at tissular oxygen tension, preferably at about 5% O₂, andlow concentration of glucose, preferably at about 1 g/l of glucose.

Another object of the invention is a kit for implementing the method ofthe invention, wherein said kit comprises means for determining ormeasuring the expression level of at least one growth factor of amesenchymal stem cells (MSC) preparation, preferably SDF-1α and/or VEGF.

In one embodiment, the expression level of at least one growth factor isassessed at the protein level, and the kit of the invention may comprisemeans for detecting the at least one growth factor, preferably SDF-1αand/or VEGF. In one embodiment, said means for detecting the at leastone growth factor is an antibody specific of said at least one growthfactor, preferably SDF-1α and/or VEGF. In one embodiment, the kit of theinvention may also comprise means for detecting the expression level ofat least one normalization protein.

In another embodiment, the expression level of at least one growthfactor is assessed at the RNA level, and the kit of the invention maycomprise means for total RNA extraction, means for reverse transcriptionof total RNA, and means for quantifying the expression of RNA of atleast one growth factor, preferably VEGF and/or SDF-1α. In oneembodiment, the means for quantifying the expression of RNA of at leastone growth factor, preferably SDF-1α and/or VEGF are PCR or qPCR primersspecific for said growth factor, preferably SDF-1α and/or VEGF. In oneembodiment, the kit also comprises reagents for carrying out aquantitative PCR (such as, for example, buffers, enzyme, and the like).In one embodiment, the kit of the invention may also comprise means fordetecting the expression level of at least one normalization gene at theRNA level.

According to one embodiment, the kit of the invention further comprisesthe reference for comparing the measured expression level of the atleast one growth factor.

In one embodiment, the kit of the invention further comprises a purefibroblasts preparation. In another embodiment, the kit of the inventionfurther comprises a pure MSC preparation.

In one embodiment, the kit of the invention comprises the supernatant ofa pure fibroblasts preparation. In another embodiment, the kit of theinvention comprises the supernatant of a pure MSC preparation. Inanother embodiment, the kit of the invention comprises a range ofdilution of pure MSC preparation supernatant and pure fibroblastspreparation supernatant. An example of dilution range is, withoutlimitation, 100/0, 75/25, 50/50, 25/75, 0/100.

The present invention also relates to a growth factor, preferably SDF-1αand/or VEGF, as a biomarker of the quality or purity of a cellpreparation comprising MSC, in particular of a cell preparationcomprising MSC to be used as MSC-based cell therapy product inregenerative medicine.

In a particular embodiment, the invention concerns a growth factor,preferably SDF-1α and/or VEGF, as a biomarker of the quality or purityof an ASC preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing ASC and DF in proliferation medium (A)and in osteogenic differentiation medium (B).

FIG. 2 is a graph showing the cell proliferation of ASC and DF accordingto the number of passages.

FIG. 3 is a histogram showing the cell survival of ASC and DF inproliferation medium without FBS, at 0.1 or 5% O₂.

FIGS. 4A-4D are a set of histograms showing KGF secretion (FIG. 4A),b-FGF secretion (FIG. 4B), IGF-1 secretion (FIG. 4C), and HGF secretion(FIG. 4D) of 5 different ASC/DF dilutions in proliferation medium with4.5 g/l glucose, at 0.1 or 5% O₂.

FIGS. 5A-5B are a set of histograms showing VEGF secretion (FIG. 5A) andSDF-1α secretion (FIG. 5B) of 5 different ASC/DF dilutions inproliferation medium with 4.5 g/l glucose, at 0.1% O₂.

FIGS. 6A-6B are a set of histograms showing VEGF secretion (FIG. 6A) andSDF-1α secretion (FIG. 6B) of 5 different ASC/DF dilutions inproliferation medium with 4.5 g/l glucose, at 5% O₂.

FIG. 7 is a histogram showing SDF-1α secretion of 5 different ASC/DFdilutions in proliferation medium with 4.5 g/l glucose, at 21% O₂.

FIGS. 8A-8B are a set of histograms showing VEGF secretion (FIG. 8A) andSDF-1α secretion (FIG. 8B) of 5 different ASC/DF dilutions inproliferation medium with 1 g/l glucose, at 0.1% O₂.

FIGS. 9A-9B are a set of histograms showing VEGF secretion (FIG. 9A) andSDF-1α secretion (FIG. 9B) of 5 different ASC/DF dilutions inproliferation medium with 1 g/l glucose, at 5% O₂.

FIG. 10 is a histogram showing SDF-1α secretion of 5 different ASC/DFdilutions in proliferation medium with 1 g/l glucose, at 21% O₂.

EXAMPLES

The present invention is further illustrated by the following examples.

Example 1 Materials and Methods

This study was performed according to the guidelines of the BelgianMinistry of Health. All procedures were approved by the EthicalCommittee of the Medical Faculty (Université Catholique de Louvain) fortissue procurement and clinical study (B40320108280). All materials wereobtained from Lonza (Verviers, Switzerland), Sigma-Aldrich (St. Louis,Mo., USA), or Invitrogen (Carlsbad, Calif., USA) unless otherwise noted.

ASC and DF Isolation and Culture

A combined harvesting of human adipose (mean: 7.4 g) and dermal (mean:1.5 cm²) tissues were performed in 8 patients (Table 1) undergoingelective plastic surgery after informed consent and serologic screening,by lipoaspiration using the Coleman technique, and skin biopsy,respectively. Adipose tissue and skin samples were kept in sterileconditions for a maximum of 60 minutes at 4° C. before adipose-derivedstem cells (ASC) and dermal fibroblasts (DF) isolation.

TABLE 1 Coupled ASC/DF donors characteristics Donor 1 2 3 4 5 6 7 8 Age19 44 40 62 56 46 45 41 (years) Sex F F F F F F F F Clinical mamma-abdomino- mamma- Lat. abdomino- mamma- mamma- mamma- indication plastyplasty plasty Dorsi plasty plasty plasty plasty flap

The adipose tissue was digested with collagenase (½ w/v) in a water bathat 37° C. for 60 minutes. Collagenase was inactivated in Dulbecco'smodified Eagle medium (DMEM) supplemented with 10% fetal bovine serum.Collected tissue was centrifuged for 10 minutes at 1500 rpm at roomtemperature. The pellet was suspended in a proliferation medium made upof DMEM supplemented with 10% fetal bovine serum, L-glutamine (2 mM),and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin, and 1μl/ml amphotericin B) and filtered through a 500-μm mesh screen. Thecollected suspension was then seeded in 25 cm² culture flasks withproliferation medium.

DF were isolated by extraction from de-epidermized dermal biopsies,minced in 2 mm×2 mm fragments and placed in plastic well. Small volumeof the proliferation medium was added to avoid detachment from theplastic surface.

After 24 hours of incubation at 37° C. and 5% CO₂, the proliferationmedia were replaced. This initial passage of the primary cells isreferred to as passage 0. Dermal pieces were removed from the culturedish when adherent cells were visible on the plastic surface surroundingtissue fragments. Cells were maintained in proliferation medium (changed2 times/week) up to passage 4, after sequential trypsinizations. Cellsfrom 3 donors were cultivated until passage 15 to study theproliferation profile in standard culture conditions (37° C., 21% O₂, 5%CO₂, 4.5 g/l glucose).

Membrane Marker Profile Characterization

At passage 4, ASC and DF were characterized for standard cell surfacemarkers (CD44, CD45, CD73, CD90, CD105, stro-1, CD106, CD146, CD166,CD19, CD31, CD11b, CD79α, CD13, HLA-DR, CD14, CD34) [Dominici et al.,Cytotherapy. 2006; 8(4):315-317; Bourin et al., Cytotherapy. 2013;15:641-648] by fluorescence-activated cell sorting (FACScan; BDBiosciences, San Jose, Calif.).

Briefly, ASC were stained with saturating amounts of monoclonalantibodies: anti-Stro-1, anti-CD90, anti-CD106, anti-CD105, anti-CD146,anti-CD166, anti-CD44, anti-CD19, anti-CD45 (Human Mesenchymal Stem Cellmarker antibody panel, R&D System, Minneapolis, Minn., USA), anti-CD44(PE mouse anti-human CD44, BD Bioscience, Franklin Lakes, N.J., USA),anti-CD73 (FITC mouse anti-human CD73, BD Bioscience), anti-CD31 (FITC,mouse anti-human, Abcam, Cambridge, UK), anti-CD11b (FITC, mouseanti-human, Abcam, Cambridge, UK), anti-CD79a (PE, mouse anti-human,Abcam, Cambridge, UK), anti-CD13 (FITC, mouse anti-human, Abcam,Cambridge, UK), anti-HLA-DR (FITC, mouse anti-human, Abcam, Cambridge,UK), anti-CD14 (FITC, mouse anti-human, Abcam, Cambridge, UK), anti-CD34(PE, mouse anti-human, Abcam, Cambridge, UK). At least 10,000 gatedevents were analyzed by flow cytometry with CellquestPro software.Results are expressed in mean fluorescence intensity (MFI), andexpressed as percentage of positive cells (threshold: 95% of isotype).

Differentiation Capacity

ASC and DF were tested at passage 4 in specific media to assess thecapacity of differentiation toward osteogenic lineage. Thedifferentiation was evaluated by Alizarin red staining after culturingthe cell during 3 weeks in specific differentiation medium(proliferation medium supplemented with dexamethasone (1 μM), sodiumascorbate (50 μg/ml), and sodium dihydrophosphate (36 mg/ml) [Qu et al.,In Vitro Cell Dev Biol Anim. 2007; 43:95-100]. Osteogenicdifferentiation was confirmed by staining for calcium phosphate withAlizarin red after formalin fixation. In addition, immunohistochemistryfor osteocalcin was performed to confirm the bone phenotype.

Impact of Oxygen Tension and Fetal Bovine Serum (FBS) on CellProliferation: EdU Assay

Cell proliferation capacity was tested by direct DNA synthesismeasurement by 5-ethynyl-2′-deoxyuridine incorporation using Click-iT®EdU Alexa Fluor® 488 Flow Cytometry Assay Kit (Life Technology, Waltham,Mass., USA). ASC (n=3) and DF (n=3) were seeded in 21.5 cm² culturedishes at a density of 5000 cells/cm², and cultured for 24 hours in 10%FBS, 21% O₂. Cells proliferation was then stopped by replacing theproliferation medium by the same, without FBS, for 24 hours. The cellswere finally placed for 48 hours in the specific conditions: 0.1% O₂, 5%O₂ and 21% O₂ in proliferation medium supplemented with 1% FBS or 5% FBSand EdU (5-ethynyl-2′-deoxyuridine, a nucleoside analog of thymidine andincorporated into DNA during active DNA synthesis) was added. Afterrevelation with Alexa Fluor® 488, positive cells were counted by flowcytometry (FACS can; BD Biosciences, San Jose, Calif.).

Growth Factor Secretion Profile

After trypsinization, cells (after passage 3) were counted and 5progressive dilutions were obtained: 100% ASC+0% DF; 75% ASC+25% DF; 50%ASC+50% DF; 25% ASC+75% DF; and 0% ASC+100% DF, and seeded in 12-wellculture plates with cells at a density leading to about 80% to 95%confluence in triplicate for incubation in hypoxic chambers (ModularIncubator Chamber MIC-101; Billups-Rothenberg, Del Mar, Calif., USA) at0.1% O₂ and 5% O₂, corresponding to highly hypoxic environment andtissular oxygen tension, respectively. The cells were exposed (for eachdilution and oxygen tension) to normoglycaemic (1 g/L) or hyperglycaemic(4.5 g/L) proliferation media. After incubation for 24 hours in thesecontrolled conditions; cell culture supernatants were harvestedindividually and stored at −20° C. for further growth factorquantification by enzyme-linked immunosorbent assay (VEGF, HGF, IGF-1,SDF-1α and basic FGF by Quantikine ELISA kit; R&D System, Minneapolis,Minn., USA). Cellular viability was assessed immediately after thehypoxic stress by3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumsolution (MTS; Promega, Leiden, the Netherlands) assay.Hypoxic/glycaemic stress tests and growth factor quantifications wereperformed in triplicate and duplicate, respectively. Results areexpressed in picograms per millimeter.

Statistical Analysis

The one-sample Kolmogorov test and Q-Q plots were used to assess thenormal distribution of values. Statistically significant differencesbetween groups (with normal distribution) were tested by paired t-testand one-way analysis of variance with the Bonferroni post hoc test.Statistical tests were performed with PASW 18 (SPSS; IBM, New York,N.Y., USA); p<0.05 was considered significant.

Results

Surface marker profiles do not allow the distinction between the twocell populations (Table 2).

TABLE 2 Surface marker characterization of human ASC and DF ASC DF % ofpositive % of positive cells cells Mesenchymal (stromal) cells markersCD13 99.06 99.86 CD44 95.53 99.97 CD73 93.78 99.86 CD90 98.63 100.00CD105 96.86 99.78 CD166 60.74 96.51 Bone marrow-derived MSC markersCD106 5.41 2.83 Stro-1 4.03 5.73 CD146 7.16 33.91 Endothelial cellsmarkers CD31 5.59 5.41 Hematopoietic lineage markers CD14 6.75 28.27CD45 5.15 0.62 CD11b 5.80 8.65 CD34 5.53 0.54 Human leukocyte antigensHLA-DR 6.52 1.65 CD19 4.51 2.05 CD79α 5.10 0.37

ASC and DF were positive (>90% of positive cells) for mesenchymal cellmarkers (CD13, CD44, CD73, CD90, CD105, CD166), negative for endothelial(CD31), bone-marrow-derived stromal cells (CD106, Stro-1, CD146) andhematopoietic markers (CD14, CD45, CD11b, CD34), and for HLA-DR, CD79aand CD19. After culture in specific differentiation media (FIG. 1),osteogenic differentiation capacity was demonstrated for both ASC and DFby Alizarin red staining and osteocalcin immunohistochemistry.

ASC and DF had similar proliferation profile until passage 15 (FIG. 2,NS).

ASC and DF viability was not significantly impacted after 24 hours ofculture at 0.1% O₂ and 5% O₂ without FBS (FIG. 3). At 5% O₂, DFviability was reduced when compared to ASC (87.04% of ASC survival,p<0.05).

The study of HGF, IGF-1, bFGF and KGF secretion (at 0.1% and 5% 02, 4.5g/l glucose) from the sequential dilutions of ASC and DF did notdemonstrate any significant curve (FIGS. 4A-4D).

However, for VEGF and SDF-1α, linear regressions following ASC“contamination” by DF were observed. Indeed, SDF-1α secretion leveldecreases with increasing ASC proportion. This result is found indifferent conditions of oxygen tension (21%, 5% or 0.1%) or of glucoseconcentrations (1 g/l or 4.5 g/l) (FIGS. 5 to 10). Moreover, in highglucose culture conditions and at 0.1% O₂ and 5% O₂ VEGF secretion levelincreases with increasing ASC proportion (FIGS. 5A and 6A). The samemeasurements in low glucose conditions demonstrated significant linearregressions for VEGF secretion at 5% O₂ (FIG. 8A).

The relations were inversed since DF release higher levels of SDF-1α andVEGF was produced in higher rates by ASC, allowing the measurement ofthe cell proportion (ASC purity).

1-14. (canceled)
 15. An in vitro method for assessing, evaluating and/ormonitoring the purity of a cell preparation comprising mesenchymal stemcells (MSC), wherein said method comprises measuring the expressionlevel of at least one growth factor expressed by said cell preparation,wherein said at least one growth factor is SDF-1α and/or VEGF.
 16. Thein vitro method according to claim 15, wherein said method furthercomprises comparing the measured expression level with a referenceexpression level.
 17. The in vitro method according to claim 15, whereinsaid mesenchymal stem cells are isolated from tissues selected from thegroup comprising adipose tissue, bone marrow, umbilical cord blood,amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopiantube, corneal stroma, lung, muscle and fetal liver.
 18. The in vitromethod according to claim 15, wherein said mesenchymal stem cells areadipose stem cells (ASC).
 19. The in vitro method according to claim 15,wherein said expression level is assessed at the protein level,preferably by the detection and/or quantification of said at least onegrowth factor secreted in the cell culture supernatant.
 20. The in vitromethod according to claim 15, wherein said expression level is assessedat the RNA level, preferably by RT-PCR, RT-qPCR, Northern Blot and/orhybridization techniques.
 21. The in vitro method according to claim 15,wherein said cell preparation is substantially pure when the SDF-1αexpression level is of at most 100 pg/ml, preferably of at most 50, 40,30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 pg/ml.
 22. The in vitro method according to claim 15, whereinsaid cell preparation is substantially pure when the SDF-1α expressionlevel is of at most 100 pg/ml, preferably of at most 50, 40, 30, 25, 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1pg/ml; and/or the VEGF expression level is of at least 200 pg/ml in thecell culture medium, preferably of at least 250, 260, 270, 280, 281,282, 283, 284, 285, 286, 287, 288, 289 or 290 pg/ml; wherein said cellpreparation is cultured in hypoxic conditions, preferably at about 0.1%O₂, and at high concentration of glucose, preferably at about 4.5 g/l ofglucose, before measuring the expression level.
 23. The in vitro methodaccording to claim 15, wherein said cell preparation is substantiallypure when the SDF-1α expression level is of at most 100 pg/ml,preferably of at most 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and/or the VEGFexpression level is of at least 90 pg/ml in the cell culture medium,preferably of at least 95, 100, 105, 110, 111, 112, 113, 114, 115, 116,117, 118, 119 or 120 pg/ml, wherein said cell preparation is cultured attissular oxygen tension, preferably at about 5% O₂, and at highconcentration of glucose, preferably at about 4.5 g/l of glucose, beforemeasuring the expression level.
 24. The in vitro method according toclaim 15, wherein said method is for assessing the quality or purity ofa cell preparation comprising mesenchymal stem cells (MSC), wherein saidcell preparation comprising MSC is to be used as MSC-based cell therapyproduct in regenerative medicine.
 25. A cell population identified by anin vitro method for assessing, evaluating and/or monitoring the purityof a cell preparation comprising mesenchymal stem cells (MSC), whereinsaid method comprises measuring the expression level of at least onegrowth factor expressed by said cell preparation, wherein said at leastone growth factor is SDF-1α and/or VEGF.
 26. A kit for implementing anin vitro method for assessing, evaluating and/or monitoring the purityof a cell preparation comprising mesenchymal stem cells (MSC), whereinsaid method comprises measuring the expression level of at least onegrowth factor expressed by said cell preparation, wherein said at leastone growth factor is SDF-1α and/or VEGF, wherein said kit comprisesmeans for determining or measuring the expression level of at least onegrowth factor, optionally further comprising the reference for comparingthe expression level of said at least one growth factor.